Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays

Abstract

The present disclosure provides methods and devices for simultaneous identification of a plurality of target nucleic acid sequences in a single sample chamber that includes an addressable array of nucleic acid probes attached to a solid surface. Addressable signals can be generated and measured, in real-time, upon hybridization of target sequences at the individual probe locations within the array while the temperature of the system is varied. Such generated signals, as a function temperature, can then be used to compute the properties of nucleic acid hybridization at each addressable location which is ultimately utilized to estimate the sequence of the target nucleic acids. In particular, an integrated semiconductor biosensor array device can be used to measure the addressable signals.

1 .- 30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . A system for assaying a presence of a target nucleic acid molecule in a sample, comprising:
 a. a chip comprising an integrated sensor adjacent to a sample chamber, wherein said sample chamber is configured to retain said sample comprising said target nucleic acid molecule, and wherein said integrated sensor comprises:
 i. a surface comprising a probe coupled to an energy donor, wherein said probe is configured to selectively couple to said target nucleic acid molecule, and, 
 ii. an optical detector below said surface, wherein said optical detector is configured to detect at least one optical signal generated upon energy transfer between an energy acceptor coupled to said target nucleic acid molecule and said energy donor, wherein said at least one optical signal is indicative of an interaction between said energy acceptor and said energy donor, and wherein said energy acceptor is configured to quench optical activity of said energy donor; 
   b. a computer processor coupled to said chip and programmed to:
 i. subject said surface to a temperature change while said sample is in said sample chamber, 
 ii. measure said at least one optical signal while said surface is subjected to said temperature change, and 
 iii. generate signal versus temperature data using a measurement of said at least one optical signal with said temperature change. 
   
     
     
         33 . The system of  claim 32 , wherein said optical detector is in an array of optical detectors below said surface. 
     
     
         34 . The system of  claim 33 , wherein said surface further comprises an additional probe coupled to an additional energy donor, wherein said array of optical detectors comprises a first optical detector and a second optical detector, wherein said first optical detector is configured to detect said at least one optical signal, and wherein said second optical detector is configured to detect at least one additional optical signal generated upon energy transfer between (1) an additional energy acceptor coupled to an additional target nucleic acid molecule of said sample and (2) said additional energy donor. 
     
     
         35 . The system of  claim 33 , wherein said array of optical detectors comprises at least about 100 integrated sensors. 
     
     
         36 . The system of  claim 33 , wherein an individual integrated sensor of said array of optical detectors is individually addressable. 
     
     
         37 . The system of  claim 32 , wherein said optical detector comprises charge-coupled device (CCD) arrays, complementary metal-oxide-semiconductor (CMOS) imagers, n-type metal-oxide semiconductor (NMOS), active-pixel sensors (APS), photomultiplier tubes (PMTs), or any combination thereof. 
     
     
         38 . The system of  claim 32 , wherein said chip further comprises a control sensor adjacent to said sample chamber, wherein said control sensor comprises a control probe that is not configured to selectively couple to said target nucleic acid molecule, and wherein said control sensor is configured to detect at least one control signal from said sample. 
     
     
         39 . The system of  claim 38 , wherein said computer processor is further programmed to (iv) measure said at least one control signal while said surface is subjected to said temperature change, and (v) normalize said signal versus temperature data against a measurement of said at least one control signal. 
     
     
         40 . The system of  claim 32 , wherein said probe is at an independently addressable location of said surface. 
     
     
         41 . The system of  claim 40 , wherein said surface comprises a plurality of probes, and wherein said plurality of probes comprises identical sequences. 
     
     
         42 . The system of  claim 40 , wherein said at least one optical signal is a discrete signal measured at said individually addressable location. 
     
     
         43 . The system of  claim 32 , wherein said computer processor is further programmed to not measure said at least one optical signal during amplification of said target nucleic acid molecule within said sample chamber. 
     
     
         44 . The system of  claim 32 , wherein said at least one optical signal is a change in signal when an amount or a rate of an interaction between said target nucleic acid molecule and said probe is changed. 
     
     
         45 . The system of  claim 32 , wherein said at least one optical signal is measured against background. 
     
     
         46 . The system of  claim 32 , wherein said computer processor is further programmed to detect a single nucleotide polymorphism (SNP) in a sequence of said target nucleic acid molecule using said signal versus temperature data. 
     
     
         47 . The system of  claim 32 , wherein said probe is an oligonucleotide. 
     
     
         48 . The system of  claim 47 , wherein a sequence of said target nucleic acid molecule is configured to form a loop structure when hybridized to said oligonucleotide. 
     
     
         49 . The system of  claim 32 , wherein said surface comprise an additional probe that is configured to selectively couple to an additional target nucleic acid molecule of said sample. 
     
     
         50 . The system of  claim 32 , wherein said computer processor is further programmed to measure said at least one optical signal while (1) said surface is subjected to said temperature change and (2) said sample is in contact with said surface. 
     
     
         51 . The system of  claim 32 , wherein said computer processor is further programmed to use said signal versus temperature data to assess a thermodynamic characteristic of an interaction between said probe and said target nucleic acid molecule.